31 research outputs found
Application of Hydrogen/Deuterium Exchange-Mass Spectrometry to Biopharmaceutical Development Requirements: Improved Sensitivity to Detection of Conformational Changes
The
usefulness of the higher-order structure information provided by hydrogen/deuterium
exchange mass spectrometry (HDX-MS) in the protein therapeutic field
is undisputed; however, its applicability as a method for critical
quality and comparability assessment has until now not been demonstrated.
Here we present results demonstrating for the first time the applicability
of the HDX-MS technique to monitor structural changes due to methionine
oxidation at sensitivity levels realistic to the requirements of biopharmaceutical
research and development. For the analyzed heavy chain marker peptides
deuterium uptake differences due to oxidation at the conserved methionine
in position 254 were significantly verifiable at the lowest increase
(1%) through spiked oxidized IgG1
Detailed Characterization of Monoclonal Antibody Receptor Interaction Using Affinity Liquid Chromatography Hyphenated to Native Mass Spectrometry
We report on the
online coupling of FcRn affinity liquid chromatography
(LC) with electrospray ionization mass spectrometry (ESI-MS) in native
conditions to study the influence of modifications on the interaction
of recombinant mAbs with the immobilized FcRn receptor domain. The
analysis conditions were designed to fit the requirements of both
affinity LC and ESI-MS. The mobile phase composition was optimized
to maintain the proteins studied in native conditions and enable sharp
pH changes in order to mimic properly IgGs Fc domain/FcRn receptor
interaction. Mobile phase components needed to be sufficiently volatile
to achieve native MS analysis. MS data demonstrated the conservation
of the pseudonative form of IgGs and allowed identification of the
separated variants. Native FcRn affinity LC–ESI-MS was performed
on a therapeutic mAb undergoing various oxidation stress. Native MS
detection was used to determine the sample oxidation level. Lower
retention was observed for mAbs oxidized variants compared to their
intact counterparts indicating decreased affinities for the receptor.
This methodology proved to be suitable to identify and quantify post-translational
modifications at native protein level in order to correlate their
influence on the binding to the FcRn receptor. Native FcRn affinity
LC–ESI-MS can tremendously reduce the time required to assess
the biological relevance of the IgG microheterogeneities thus providing
valuable information for biopharmaceutical research and development
SPR analysis on FcγRI (A), FcγRIIa (B), and FcγRIIIa binding (C) in ascending intensity.
<p>Bulk material is used as reference and set to 100%. All samples are measured in triplicates.</p
Effect of Fc glycoengineering on ADCC activity.
<p>The ADCC activities are quantified relative to a reference material set to 100% by full curve parallel line analysis. For each sample, the box plot represents 5 independent measures of duplicates. The dashed lines indicate the 95% confidence interval (CI) of the bulk material.</p
Chemo-Enzymatic Synthesis of <sup>13</sup>C Labeled Complex N‑Glycans As Internal Standards for the Absolute Glycan Quantification by Mass Spectrometry
Methods for the absolute quantification
of glycans are needed in
glycoproteomics, during development and production of biopharmaceuticals
and for the clinical analysis of glycan disease markers. Here we present
a strategy for the chemo-enzymatic synthesis of <sup>13</sup>C labeled
N-glycan libraries and provide an example for their use as internal
standards in the profiling and absolute quantification of mAb glycans
by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF)
mass spectrometry. A synthetic biantennary glycan precursor was <sup>13</sup>C-labeled on all four amino sugar residues and enzymatically
derivatized to produce a library of 15 glycan isotopologues with a
mass increment of 8 Da over the natural products. Asymmetrically elongated
glycans were accessible by performing enzymatic reactions on partially
protected UV-absorbing intermediates, subsequent fractionation by
preparative HPLC, and final hydrogenation. Using a preformulated mixture
of eight internal standards, we quantified the glycans in a monoclonal
therapeutic antibody with excellent precision and speed
Production workflow for the different glycan variants of IgG1.
<p>Numbers in parenthesis represent the days needed for sample preparation. Starting material denoted as “bulk” is material obtained from the production process after regular fermentation and purification steps.</p
Summary Table—Impact of increasing galactose / sialic acid levels of IgG1 on FcγRI, IIa, and IIIa binding and ADCC activity.
<p>Summary Table—Impact of increasing galactose / sialic acid levels of IgG1 on FcγRI, IIa, and IIIa binding and ADCC activity.</p
Chemo-Enzymatic Synthesis of <sup>13</sup>C Labeled Complex N‑Glycans As Internal Standards for the Absolute Glycan Quantification by Mass Spectrometry
Methods for the absolute quantification
of glycans are needed in
glycoproteomics, during development and production of biopharmaceuticals
and for the clinical analysis of glycan disease markers. Here we present
a strategy for the chemo-enzymatic synthesis of <sup>13</sup>C labeled
N-glycan libraries and provide an example for their use as internal
standards in the profiling and absolute quantification of mAb glycans
by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF)
mass spectrometry. A synthetic biantennary glycan precursor was <sup>13</sup>C-labeled on all four amino sugar residues and enzymatically
derivatized to produce a library of 15 glycan isotopologues with a
mass increment of 8 Da over the natural products. Asymmetrically elongated
glycans were accessible by performing enzymatic reactions on partially
protected UV-absorbing intermediates, subsequent fractionation by
preparative HPLC, and final hydrogenation. Using a preformulated mixture
of eight internal standards, we quantified the glycans in a monoclonal
therapeutic antibody with excellent precision and speed
FcγRIIIa column assay analysis.
<p>Normalized UV chromatograms of the hypo- and hyper-galactosylated as well as the mono-sialylated samples are exemplarily shown (A). UV absorbance was measured at 280 nm. Retention times for all batches are compared for the fucosylated (early eluting) peak (B) and the partly/non-fucosylated (late eluting) peak (C).</p
Relative quantification of HER2 methionine oxidation (Met-83, Met-107, and Met-255) by ion current chromatogram analysis of the oxidized product and its parent peptide using the quantification software GRAMS/32™.
<p>Relative quantification of HER2 methionine oxidation (Met-83, Met-107, and Met-255) by ion current chromatogram analysis of the oxidized product and its parent peptide using the quantification software GRAMS/32™.</p